Exosome介导的lincRNA-HOTAIR和PTEN互为ceRNA调控喉癌凋亡与自噬的机制研究

Recently proposed hypothesis of “competing endogenous RNA (ceRNA)” has attributted a new, global function for all the non-coding RNAs. However, the biological relevance of the ceRNA hypothesis has not been investigated in apoptosis and autophagy of laryngeal squamous cell carcinomas (LSCC). We previously demonstrated that exosomal lincRNA HOTAIR，PTEN and several important MiRNAs were significantly higher in patients with LSCC than those with vocal cord polyps. There was a significant correlation between exosomal HOTAIR expressions and the lymph node metastasis, advanced T classifications (T3/T4) or clinical stages (III/IV). In a separate study, we found that siRNAmediated knockdown of HOTAIR led to reduced invasion and increased apoptosis of Hep-2 cells in vitro and significantly reduced growth of LSCC xenograft tumors in mice. Moreover, PTEN methylation was significantly reduced in Hep-2 cells depleted of HOTAIR. These findings, together our previous studies on miRNAs, stimulate us to explore the synergistic regulation mechanism and network regulatory function of lincRNA-miRNA-mRNA in LSCC. By Bioinformatics Analysis Approach, HOTAIR and PTEN were identified to regulate each other by a ceRNA. Thus, we propose that exosome mediated HOTAIR and PTEN can regulate apoptosis and autophagy in LSCC by a cross talk through their ability to compete for a ceRNA. By utilizing the techniques of molecular cloning,reporter gene,qRT-PCR and so on, we would scrutinize mechanistically the functions of exosomal HOTAIR and PTEN in ceRNA network for the regulation of apoptosis and autophagy in LSCC and by identifying the binding sites for miRNAs, find the correlation between functional molecules of lincRNA-miRNA-mRNA and apoptotic or autophagic regulatory pathways. Finally, a single unit (oligonucleotide, ODN) that is capable of simultaneously expressing one or more target genes will be engineered to investigate the possibility and effectiveness of the inhibition for invasion and metastasis of LSCC. The main objectives of this research are to demonstrate the mechanisms of apoptosis and autophagy regulated by a ceRNA hypothesis and the impact of the cross talk between exosomal HOTAIR and PTEN on tumorigenesis of LSCC, demonstrate the efficiency of a single sequence targeting multi genes in suppressing the cancer invasive properies, which will be essential for developing improved treatment plans for LSCC.

“竞争性内源RNA（competing endogenous RNA，ceRNA）”假说揭示了一种RNA间相互作用的新机制。然而，ceRNA在喉癌发生发展的作用机制尚不清楚。我们前期发现喉癌细胞含有丰富的微囊泡（exosome），且不同程度地表达lincRNA HOTAIR、PTEN以及多个miRNA，使申请人联想到lincRNA-miRNA-mRNA的协同调控关系是否在喉癌中发挥作用。经预测，HOTAIR与PTEN基因可以通过ceRNA机制进行相互调控，因此，课题组提出“微囊泡介导的 HOTAIR与PTEN互为ceRNA调控喉癌凋亡和自噬”的科学假说。本课题将从体内外水平揭示ceRNA调控网络中exosome介导的HOTAIR和PTEN调控喉癌凋亡和自噬的新机制，并针对性地设计相应反义或拟核苷酸制剂，探讨抑制喉癌发生发展的可能性及有效性。该课题的完成将为建立喉癌新的分子治疗策略提供基础。

喉癌是头颈部常见的恶性肿瘤之一，近年来随着综合治疗的不断进展，喉癌患者的死亡率有所下降，但是生存率仍然约为50%。因此如何提高患者的生存率，挖掘喉癌分子机制仍然是当下急需攻克的课题，寻找新的有效的治疗靶点成为研究的关键问题。微囊泡（MVs）作为细胞间信息的重要介质，运载与肿瘤的侵袭和转移密切相关的非编码RNA(ncRNAs)，大量的研究表明多种ncRNA在喉癌的发生发展中扮演重要角色。本项目在执行期间从三方面进行相关研究：1、我们首先比较分析联合miR-375、206可以抑制喉癌细胞增殖和侵袭以及诱导其凋亡。同时我们还证实miR-26b可以增加喉癌的化疗敏感性；2、竞争性内源RNA调控网络的基础是在RNA 组学的基础上构建的庞大的调控网络，我们分析构建了多个喉癌相关ceRNA网络，包括特异性炎性基因相关的ceRNA网络与ncRNA介导的ceRNA网络。3、我们成功提取了微囊泡，并通过电镜以及Western进行了证实，验证了circRASSF2 作为ceRNA分子竞争性吸附mir-302b-3p调控IGF-1R促进喉癌进展的科学假设。依托本基金参与资助的研究工作所取得的相关数据，共发表相关SCI文章5篇，该课题的结果可以为ceRNA研究领域提供全新信息，为喉癌生物靶向治疗提供新的思路，为建立新的靶向治疗策略提供理论基础，并且本项目中所涉及的临床研究成果可以进一步在临床中起到应用价值。